Optical Imaging Paves the Way for Autophagy Research

Abstract

Autophagy is a degradation process in eukaryotic cells that recycles cellular components for nutrition supply under environmental stress and plays a double-edged role in development of major human diseases. Noninvasive optical imaging enables us to clearly visualize various classes of structures involved in autophagy at macroscopic and microscopic dynamic levels. In this review, we discuss important trends of emerging optical imaging technologies used to explore autophagy and provide insights into the mechanistic investigation and structural study of autophagy in mammalian cells. Some exciting new prospects and future research directions regarding optical imaging techniques in this field are also highlighted.

Keywords: autophagy; fluorescent probe; optical imaging; super-resolution.

Figure 1

Autophagy-Related Marker Proteins. A few protein complexes that specially accumulate in different stages of autophagy can be employed in optical imaging to indicate the autophagic flux. At the beginning of autophagy, a phagophore originating from the phagophore assembly site (PAS) with initial complexes containing ULK and PI₃K Class III (also known as omegasomes) elongates and seals itself around the selected cargoes to form a double-membrane structure called an autophagosome. The autophagosome then fuses with the lysosome individually, or alternatively, sequentially with the late endosomes and lysosome (not shown), to generate an autolysosome. After a degradation process, cytoplasm or other various unwanted cellular components and invading microbes can be recycled and hydrolyzed into amino acids, glucose, and lipids, which then return back to the cytoplasm for energy supply and biomacromolecule synthesis to maintain cellular homeostasis. The marker proteins at the different stages of autophagy can be labeled by bioprobes as potential indicators of autophagic intensity or the whole autophagic flux. LAMP, lysosome-associated membrane protein; MDC, monodansylcadaverine; PI3K, phosphoinositide 3-kinase; ULK, Unc-51-like kinase.

Figure 2

Milestones of Optical Imaging in Autophagy Research. These events involve specific fluorescence probing methods that localize the selected autophagic components and optical imaging platforms. Relative to the half-century history in the autophagy field, most of the new investigative methods and advanced imaging equipment were developed during the past decade, during which some super-resolution optical imaging technologies have emerged. CLEM, correlated light and electron microscopy; dSTORM, direct stochastic optical reconstruction microscopy; LC3; light chain 3; LSCM, laser scanning confocal microscopy; SIM, structured illumination microscope; STED, stimulated emission depletion microscopy; TIRFM; total internal reflection fluorescence microscopy.

Figure 3

Key Figure: Developmental Routines of Fluorescent Imaging Probes and standardizing patterns of optical imaging procedures for assessment of autophagy were schematized for researchers. To investigate the target of interest (purple ring), an elaborately designed fluorescence probe (green star) is used to tag the targets, which involve initial complexes, phagophore, autophagosome, autolysosome, lysosome, and other related autophagic cargoes in an ordinarily defined autophagic machine. Five mature fluorescent probing methods – fluorescent protein tags (e.g., reporter gene transfection and expression), luciferase reporter system (bioluminescence), organic dyes (e.g., small-molecule fluorophores), fluorescent nanoparticles (e.g., AIE and quantum dot nanomaterials), and fluorescently labeled antibodies (e.g., direct/indirect immunofluorescence assay) – are candidates to observe these structures by optical imaging. Obviously, the scales of the different autophagic compartments (usually ranging from 0.1 to 1 μm), the specificity of the probing methods, and the characteristics of specimen (species, thickness, and live), as well as the experimental objective that one intends to achieve, may impact the selection of a suitable optical imaging instrumentation for a researcher to visualize the different stages of autophagy or the whole autophagic flux. AIE, aggregation-induced emission.

Figure 4

Multicolor Fluorescence Imaging (A) and an Autophagy Reporter (B). In a representative fluorescence colocalization imaging experiment, the reference component (Part a) is known for the label of Probe 1, and the target of interest (Part b) is unknown but is labeled with Probe 2. When these two parts encounter each other, three possible situations including colocalization, non-colocalization, or partial colocalization may occur, which generates the merged color of Probe 1 and Probe 2 (pink); individual color of Probe 1 (red) and Probe 2 (blue); or mixed colors of red, blue, and pink, respectively. The working process interpretation of a tandem LC3 fluorescence protein reporter gene delivery system (RFP–GFP–LC3) for autophagic flux evaluation is shown in (B). In the tandem double RFP–GFP–LC3 system, the two colors of GFP and RFP overlay and exhibit yellow in autophagosome. However, GFP is acid sensitive and is fluorescently quenched by the acid hydrolases of the lysosome, which join in the autophagosome to form an autolysosome, resulting in a monochromatic fluorescence of RFP. Thus, the autophagosome and autolysosome can be specifically labeled with yellow and red, respectively. Using this novel fluorescence protein probe, the autophagic flux can be easily traced under different kinds of fluorescence microscopes. LC3, light chain 3; RFP, red fluorescent protein.